The activation of pro-phenol oxidase is an important physiological process involved in defense mechanisms in insects. These include: cuticle sclerotization, wound healing, encapsulation and melanization of foreign materials, and generation of cytotoxic molecules. Knowledge of the activation process from model insects such as Manduca sexta will be useful for studying similar systems in blood-sucking insects that transmit human, diseases. Goals of this research are to characterize the pro-phenol oxidase activating proteinase and to study mechanisms that regulate the enzyme in M. sexta. The specific aims of the project are: 1. Purification and characterization of the pro-phenol oxidase activating proteinase. The activating proteinase will be purified from both hemolymph and cuticle. Amino acid sequence and other biochemical characteristics of the enzyme will be studied. Enzymatic properties of the proteinase with and without a cofactor, named factor A, will be investigated to understand the activation mechanism of pro-phenol oxidase. 2. Molecular cloning and expression of the pro-phenol oxidase activating proteinase. With the amino acid sequence information and antiserum to the activating proteinase available, its cDNA will be cloned by screening M. sexta hemocyte, fat body or epidermis cDNA libraries. The proteinase will be expressed as a recombinant protein in its zymogen form for a study of upstream activators and eventual reconstitution of the pro-phenol oxidase activating system. Site, time, level, and inducibility of expression of the enzyme in M. sexta will be investigated. 3. Regulation of the pro-phenol oxidase activating proteinase by serine proteinase inhibitors. The inhibition reactions of the proteinase by M. sexta serpins and Kunitz-type trypsin inhibitors will be investigated to understand the physiological regulation of the enzyme. 4. Purification and characterization of factor A. Factor A which alters the activity and specificity of pro-phenol oxidase activating proteinase will be purified from cuticle and hemolymph. Its interactions with the activating proteinase and pro-phenol oxidase will be characterized. This research will contribute to our understanding of the biochemical process of pro-phenol oxidase activation, and will generate useful molecular probes for studying similar systems in insect vectors of human diseases.